Hepatocellular carcinoma specific promoter and uses thereof

ABSTRACT

Liver cancer, particularly hepatocellular carcinoma specific promoter sequences and adenovirus vehicles are provided. By providing for transcriptional initiating regulation dependent upon transcription factors that are only active in specific, limited cell types, virus replication will be restricted to the target cells. The modified adenovirus may be used as a vehicle for introducing new genetic capability, particularly associated with cytotoxicity, for treating neoplasia.

BACKGROUND OF THE INVENTION

Cancer of the liver is a rare malignancy in the United States, but inparts of Asia and Africa, it is one of the most common malignancies. Theareas of highest incidence are in eastern Asia and sub-Saharan Africa,and HCC can be the dominant cause of cancer mortality in these areas.Although incidence is very low in Western countries, ethnic subgroups inthese populations may be at very high risk due to the high prevalence ofchronic hepatitis B virus (HBV) infection. Because survival times forpatients with HCC are short, incidence and mortality figures are roughlyequivalent. In the United States, 5-year survival rates for livercancers over the past two decades remain at about 5%.

The epidemiologic association of chronic HBV or HCV infection withhepatocellular carcinoma has been well established. For patients withchronic viral hepatitis, screening for early-stage hepatocellularcarcinoma may permit the institution of curative treatment strategies,and antiviral treatment may reduce the risk of subsequent development ofhepatocellular carcinoma. For patients with established hepatocellularcarcinoma, the presence of concurrent chronic viral hepatitis orcirrhosis may affect prognosis and survival and may alter treatmentoptions because of impaired hepatic function.

Incidence of hepatocellular carcinoma increases with age, with the ageof peak incidence varying somewhat with population. HCC is seen inchildhood in areas where HBV is endemic and infection occurs primarilyat birth or early childhood. In all populations worldwide, there is astrong male predominance in HCC incidence. Several possible explanationsfor this have been proposed, including higher rates of HBV carriageamong males, genetic susceptibility, androgenic steroids, higher bodyiron stores, and higher exposure to other hepatocarcinogenic cofactors(e.g., alcohol, aflatoxin).

Familial aggregation of HCC has been reported, primarily in familieswhere chronic HBV infection is also present. Some studies, however, havefound an effect of family history independent of HBV status. Familialaggregation may be explained by the interaction of HBV and a major gene.Familial aggregation has also been reported for hepatoblastoma, whichoccurs in families carrying germline mutations of the APC (adenomatouspolyposis coli) gene.

Because of the high prevalence of HBV infection in certain regions ofChina, a screening program for hepatocellular carcinoma was institutedfor adults over the age of 35 with chronic HBV infection. The screeningtests used were serum alpha-fetoprotein (AFP) and liver ultrasonographyperformed every 6 months. The AFP has a sensitivity of 70%, since up to30% of hepatocellular carcinomas do not secrete it. Liverultrasonography has up to 70% sensitivity for the detection of HCCs thatare less than 2 cm, but has poor specificity. The combination of AFP andultrasonography, however, increases both sensitivity and specificity.

The prognosis for liver carcinoma patients is poor. Patients withmetastatic or locally advanced HCC usually respond poorly to anticancertreatments. Untreated patients usually die in 3-4 months; treatedpatients may live 6 to 18 months if they respond to therapy. Long-termsurvival is seen occasionally after successful subtotal hepatectomy fornoninvasive carcinoma. Because the normal metabolic and storagefunctions of the liver are impaired, patients are at risk fornutritional and bleeding complications. Patients with advanced cirrhosiscommonly succumb to complications such as encephalopathy, varicealhemorrhage, and sepsis, independently of the tumor's extent.

Treatment decisions are also based on the presence of active hepatitisor cirrhosis. Doxorubicin, the most active chemotherapy agent for HCC,is metabolized and excreted by the liver. The pharmacokinetics fordoxorubicin may be changed for patients with liver dysfunction,resulting in enhanced toxicity. Hepatic resection, a treatment of choicefor solitary HCC, can result in hepatic failure if hepatic reserve iscompromised by hepatitis or cirrhosis. For patients with unresectableHCC, orthotopic liver transplantations have produced prolonged survival.Patients with stage I or II HCC have 5-year survivals followingtransplantation that are comparable to those of patients transplantedfor cirrhosis without HCC. This survival advantage is attenuated,however, in patients with active HBV viral replication (HBeAg or highlevels of HBV DNA) because of the high incidence of severe viralre-infections of the liver allograft. Although patients with HCVinfection also have a high incidence of re-infection followingtransplantation, the disease is indolent and eventual cirrhosis may notoccur for decades.

Several viruses have recently come forth as both vehicles for genetherapy and as candidate anticancer agents. Among them adenovirus, amildly pathogenic human virus that propagates prolifically in epithelialcells, the origin of many human cancers. Adenovirus has emerged as avirus that can be engineered with oncotropic properties. See, forexample, U.S. Pat. No. 5,846,945 (Onyx); U.S. Pat. No. 5,801,029 (Onyx);U.S. Pat. No. 5,747,469 (Univ Texas); PCTUS1999/08592 (WO 99/59604;Onyx) or PCT/US1998/03514 (WO 98/35554; Canji); PCT/US1997/22036 (WO98/29555; Onyx). Replication competent adenovirus vectors have beendesigned to selectively replicate in tumor cells. Improving the deliveryof these adenoviruses, both to local-regional and disseminated disease,as well as improving the virus to promote intratumoral spread are ofparticular interest.

Several experimental cancer therapies utilize various aspects ofadenovirus or adenovirus vectors. See, for example, U.S. Pat. Nos.5,846,945; 5,801,029; PCT/US99/08592; U.S. Pat. No. 5,747,469;PCT/US98/03514; and PCT/US97/22036.

Although replication competent adenoviruses may be able to achieveselective targeting and amplification for the treatment of local anddisseminated cancer, there remains a need for improvement in both theadenovirus vectors themselves and methods for their use. Preliminaryresults suggest that the features of effective treatment strategies forvarious types of cancer may require development of specific adenovirusvectors and/or methods particular to the type of cancer under treatment.Although chemotherapy and immunotherapy are the most prevalent currenttherapeutic strategies for disseminated tumors, both toxic side effectsand lack of efficacy remain a problem.

There is, therefore, substantial interest in development of viralvectors which enable the targeting of specific cancers in vivo.

SUMMARY OF THE INVENTION

The present invention provides replication-competent adenoviral vectorscomprising a liver cancer-specific transcriptional regulatory element(TRE) operably linked to a gene required for virus replication.

In one aspect of the invention, genetic sequences comprising livercancer specific TREs derived from the CRG-L2 gene are provided. Thehepatocellular carcinoma specific TRE may be derived from the 0.8 kbsequence upstream of the translational start codon for the CRG-L2 gene,presented herein as SEQ ID NO:1, or from a 0.7 kb sequence containedwithin SEQ ID NO:1 (residues 119-803); or as an EcoRI to NcoI fragmentderived from the 0.8 kb sequence.

In one embodiment of the invention, a vector comprising the CRG-L2promoter is provided. The vector may comprise a coding sequence operablylinked to the CRG-L2 promoter. Vectors of interest include plasmids,viral vectors, genome integrating vectors, and the like. Viral vectorsof interest include adenoviral vectors.

Adenoviral vectors of the invention may comprise one or more livercancer-specific TREs, and may further comprise one or more regulatorysequences, e.g. enhancers, promoters, transcription factor binding sitesand the like, which may be derived from the same or different genes.

The adenovirus vectors may comprise co-transcribed first and secondadenoviral genes under control of a liver cancer-specific TRE and thesecond gene may be under translational control of an internal ribosomeentry site (IRES). Methods are provided for introducing into a cell anadenoviral vector comprising a liver cancer-specific TRE operably linkedto a gene required for virus replication, and host cells comprising theadenovirus vector(s). In another aspect, methods are provided forconferring selective cytotoxicity in target primary liver cancer cells,particularly hepatocellular carcinoma cells, comprising contacting thecells with an adenovirus vector of the invention, whereby the vectorenters the cell and propagates virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Sequence of CRG-L2 0.8 Kb TRE DNA.

FIG. 2. CP1505 luciferase reporter construct containing the CRG-L2 0.7Kb TRE DNA sequence.

FIG. 3. Reporter gene assay to evaluate promoter activity in hepatomacells. CRG-L2 promoter activity in Hep3B and HuH7 HCC cell lines is ≧100fold higher than pGL2 negative control. Since both cell lines are AFPpositive, AFP promoter was used as a positive control.

FIG. 4: CRG-L2 promoter expression in Hep3B, HuH7 and HBL-100 cells. Theexpression of CRG-L2 in HBL-100 cells is 8-10 fold less than that inHep3B and HuH7 cells, suggesting cell specificity.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The hepatocellular carcinoma-specific replication-competent adenovirusvectors of the invention comprise an adenovirus gene essential forreplication, preferably an early gene, under the transcriptional controlof a liver cancer-specific transcriptional regulatory element (TRE),preferably a CRG-L2 regulatory sequence. By providing one or more livercancer-specific TREs, the adenovirus vectors effect selectivereplication and corresponding cytotoxicity in primary liver cancercells.

The adenoviral vector comprising a liver cancer-specific TRE may furthercomprise one or more regulatory sequences, e.g. enhancers, promoters,transcription factor binding sites and the like, which may be derivedfrom the same or different genes. The adenovirus vectors may compriseco-transcribed first and second genes under control of a livercancer-specific TRE, wherein the second gene may be under translationalcontrol of an internal ribosome entry site (IRES). In some cases, theadenovirus vectors comprise more than two co-transcribed genes undercontrol of a liver cancer-specific TRE, wherein one or more genes isunder translational control of an internal ribosome entry site (IRES).The adenovirus vectors of the invention may or may not comprise theadenoviral E3 gene, an E3 sequence, or a portion thereof.

In another aspect, methods are provided for conferring selectivecytotoxicity in target primary liver cancer cells, particularlyhepatocellular carcinoma cells, comprising contacting the cells with anadenovirus vector of the invention, whereby the vector enters the celland propagates virus. The replication of virus in primary liver cancercells, as compared to non-tumor cells, or to normal, i.e.non-transformed cells, is usually about 10 fold greater, and may beabout 100 fold greater, and in some instances is as much as about 1000fold or more greater. The administration of virus may be combined withadditional treatment(s) appropriate to the particular disease, e.g.antiviral therapy, chemotherapy, surgery, radiation therapy orimmunotherapy. In some embodiments, this treatment suppresses tumorgrowth, e.g. by killing tumor cells. In other embodiments, the sizeand/or extent of a tumor is reduced, or its development delayed.Cytotoxicity is a term well understood in the art and refers to a statein which a cell's usual biochemical or biological activities arecompromised (i.e., inhibited), including cell death and/or cytolysis.These activities include, but are not limited to, metabolism; cellularreplication; DNA replication; transcription; translation; uptake ofmolecules. Assays known in the art as indicators of cytotoxicity,include dye exclusion, ³H-thymidine uptake, and plaque assays.

Individuals suitable for treatment by these methods include individualswho have or are suspected of having liver cancer, particularlyhepatocellular carcinoma, including individuals in the early or latestages of the disease, as well as individuals who have previously beentreated (e.g., are in the adjuvant setting). Other individuals suitablefor the methods described herein are those who are considered high riskfor developing liver cancer, such as those who are positive forinfection with a hepatitis virus, which may include hepatitis B virus;and hepatitis C virus; individuals suffering from cirrhosis; etc.Treatment regimes include both the eradication of tumors or other formsof the disease, antiviral treatment, supportive treatment for liverfunction, as well as palliation of the disease. The presence of primaryliver cancer and the suitability of the individual for receiving themethods described herein may be determined by any of the techniquesknown in the art, including diagnostic methods such as imagingtechniques, analysis of serum tumor markers, and biopsy.

The invention provides novel regulatory sequences that provide forenhanced expression in hepatocellular carcinoma cells, set forth in SEQID NO:1. This region of DNA contains the native promoter elements thatdirect expression of the linked CRG-L2 gene.

The various methods of the invention will be described below. Althoughparticular methods of tumor suppression are exemplified in thediscussion below, it is understood that any of a number of alternativemethods, including those described above are equally applicable andsuitable for use in practicing the invention. It will also be understoodthat an evaluation of the vectors and methods of the invention may becarried out using procedures standard in the art, including thediagnostic and assessment methods described above.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the scope of those of skill in the art.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Handbook of Experimental Immunology” (D. M.Weir & C. C. Blackwell, eds.); “Gene Transfer Vectors for MammalianCells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols inMolecular Biology” (F. M. Ausubel et al., eds., 1987); “PCR: ThePolymerase Chain Reaction”, (Mullis et al., eds., 1994); and “CurrentProtocols in Immunology” (J. E. Coligan et al., eds., 1991).

For techniques related to adenovirus, see, inter alia, Feigner andRingold (1989) Nature 337:387-388; Berkner and Sharp (1983) Nucl. AcidsRes. 11:6003-6020; Graham (1984) EMBO J. 3:2917-2922; Bett et al. (1993)J. Virology 67:5911-5921; Bett et al. (1994) Proc. Natl. Acad. Sci. USA91:8802-8806.

Definitions

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art and the practice of the presentinvention will employ, conventional techniques of microbiology andrecombinant DNA technology, which are within the knowledge of those ofskill of the art.

As used herein, the terms “neoplastic cells”, “neoplasia”, “tumor”,“tumor cells”, “cancer” and “cancer cells”, (used interchangeably) referto cells which exhibit relatively autonomous growth, so that theyexhibit an aberrant growth phenotype characterized by a significant lossof control of cell proliferation. Neoplastic cells can be malignant orbenign.

Hepatocellular carcinoma is a tumor that is relatively uncommon in theUnited States, although its incidence is rising, principally in relationto the spread of hepatitis C infection. It is the most common cancer insome other parts of the world, with more than one million new casesdiagnosed each year. Hepatocellular carcinoma is potentially curable bysurgical resection, but only for the small fraction of patients withlocalized disease. Prognosis depends on the degree of local tumorreplacement and the extent of liver function impairment. Therapy otherthan surgical resection is often experimental in nature, includingsystemic or infusional chemotherapy, hepatic artery ligation orembolization, percutaneous ethanol injection, radiofrequency ablation,cryotherapy, and radiolabeled antibodies, often in conjunction withsurgical resection and/or radiation therapy. A few patients may becandidates for liver transplantation, but the limited availability oflivers for transplantation restricts the use of this approach.Hepatocellular carcinoma can coexist with bile duct cancer(cholangiocarcinoma).

Hepatocellular carcinoma is associated with cirrhosis in 50% to 80% ofpatients; 5% of cirrhotic patients eventually develop hepatocellularcancer, which is often multifocal. Hepatitis B infection and hepatitis Cinfection are the most significant causes of hepatocellular carcinomaworldwide, particularly in patients with continuing antigenemia and inthose who have chronic active hepatitis. Aflatoxin has also beenimplicated as a factor in the etiology of primary liver cancer in partsof the world where this mycotoxin occurs in high levels in ingestedfood. Workers who were exposed to vinyl chloride before controls onvinyl chloride dust were instituted developed sarcomas in the liver,most commonly angiosarcomas.

The primary symptoms of hepatocellular carcinoma are those of a hepaticmass. Among patients with underlying cirrhotic disease, a progressiveincrease in alpha-fetoprotein (AFP) and/or in alkaline phosphatase or arapid deterioration of hepatic function may be the only clue to thepresence of the neoplasm. Infrequently, patients with this disease havepolycythemia, hypoglycemia, hypercalcemia, or dysfibrinogenemia.

The biologic marker AFP is useful for the diagnosis of this neoplasm. Bya radioimmunoassay technique, 50% to 70% of patients in the UnitedStates who have hepatocellular carcinoma have elevated levels of AFP.AFP levels have been shown to be prognostically important, with themedian survival of AFP-negative patients significantly longer than thatof AFP-positive patients. Other prognostic variables include performancestatus, liver functions, and the presence or absence of cirrhosis andits severity in relation to the Child-Pugh classification.

As used herein, “suppressing tumor growth” refers to reducing the rateof growth of a tumor, halting tumor growth completely, causing aregression in the size of an existing tumor, eradicating an existingtumor and/or preventing the occurrence of additional tumors upontreatment with the compositions, kits or methods of the presentinvention. “Suppressing” tumor growth indicates a growth state that iscurtailed when compared to growth without contact with, i.e.,transfection by, an adenoviral vector combined with administration ofchemotherapeutic agents and radiation as described herein. Tumor cellgrowth can be assessed by any means known in the art, including, but notlimited to, measuring tumor size, determining whether tumor cells areproliferating using a ³H-thymidine incorporation assay, or countingtumor cells. “Suppressing” tumor cell growth means any or all of thefollowing states: slowing, delaying, and stopping tumor growth, as wellas tumor shrinkage.

“Delaying development” of a tumor means to defer, hinder, slow, retard,stabilize, and/or postpone development of the disease. This delay can beof varying lengths of time, depending on the history of the diseaseand/or individual being treated.

As used herein, a “transcription response element” or “transcriptionalregulatory element”, or “TRE” is a polynucleotide sequence, preferably aDNA sequence, comprising one or more enhancer(s) and/or promoter(s)and/or promoter elements such as a transcriptional regulatory proteinresponse sequence or sequences, which increases transcription of anoperably linked polynucleotide sequence in a host cell that allows thatTRE to function. A “hepatocellular carcinoma-specific transcriptionalresponse element” preferentially directs gene expression inhepatocellular carcinoma cells. “Under transcriptional control” is aterm well understood in the art and indicates that transcription of apolynucleotide sequence, usually a DNA sequence, depends on its beingoperably (operatively) linked to an element which contributes to theinitiation of, or promotes, transcription. “Operably linked” refers to ajuxtaposition wherein the elements are in an arrangement allowing themto function.

In addition to the hepatocellular carcinoma specific TRE, the vectors ofthe invention may further comprise promoters and/or enhancers derivedfrom the same or different genes. Such additional regulatory elementsmay be operably linked to adenovirus genes essential for replication.

The term “composite TRE” refers to a TRE that comprises transcriptionalregulatory elements that are not naturally found together, usuallyproviding a non-native combination of promoters and enhancer, forexample, a heterologous combination of promoter and enhancer and/ortranscription factor binding sites; a combination of human and mousepromoter and enhancer; two or more enhancers in combination with apromoter; multimers of the foregoing; and the like. At least one of thepromoter, enhancer or and/or transcription factor binding site elementswill be hepatocellular carcinoma specific, for example the CRG-L2promoter in combination with an enhancer. In other embodiments, two ormore of the elements will provide hepatocellular carcinoma specificity.A composite TRE comprising regulatory elements from two or more sourcesmay be used to regulate one or more adenovirus genes.

Alternatively, separate TREs may be operably linked to differentadenoviral genes. For example, one TRE may regulate transcription of anadenovirus E1A gene, while a different TRE regulates expression of anadenovirus E1B gene. Additional TREs may also be operably linked totherapeutic sequences targeted for expression.

Without limitation, regulatory elements of interest for use in thepresent vectors may include the E2F promoter, the telomerase promoter,the H19 promoter (Brannan et al., Mol. and Cell. Biol., 10(1) 28-36,1990; U.S. Pat. Nos. 5,955,273; 6,306,833), and the AFP promoter. Alsoof interest are promoter elements reported to be modified in HCC,including the promoters for cyclin-dependent kinase inhibitor 4a;caspase 8, apoptosis-related cysteine protease; ras association(RaIGDS/AF-6) domain family 1 protein isoform 1 a; and cadherin 13,H-cadherin (see Yu et al. (2002) BMC Cancer 2:29).

By “transcriptional activation” or an “increase in transcription,” it isintended that transcription is increased above basal levels in a normal,i.e. non-transformed cell by at least about 2 fold, preferably at leastabout 5 fold, preferably at least about 10 fold, more preferably atleast about 20 fold, more preferably at least about 50 fold, morepreferably at least about 100 fold, more preferably at least about 200fold, even more preferably at least about 400 fold to about 500 fold,even more preferably at least about 1000 fold. Basal levels aregenerally the level of activity (if any) in a non-target cell (i.e., adifferent cell type), or the level of activity (if any) of a reporterconstruct lacking a hepatocellular carcinoma-specific TRE as tested in atarget cell line. When the TRE controls a gene necessary for viralreplication, the replication of virus is significantly higher in thetarget cells, as compared to a control cell, usually at least about2-fold higher, preferably, at least about 5-fold higher, morepreferably, at least about 10-fold higher, still more preferably atleast about 50-fold higher, even more preferably at least about 100-foldhigher, still more preferably at least about 400- to 500-fold higher,still more preferably at least about 100-fold higher, most preferably atleast about 1×10⁶ higher. Most preferably, the adenovirus replicatessolely in the target cells (that is, does not replicate or replicates ata very low levels in non-target cells).

Activity of a TRE can be determined, for example, as follows. A TREpolynucleotide sequence or set of such sequences can be generated usingmethods known in the art, such as chemical synthesis, site-directedmutagenesis, PCR, and/or recombinant methods. The sequence(s) to betested can be inserted into a vector containing a promoter (if nopromoter element is present in the TRE) and an appropriate reporter geneencoding a reporter protein, including, but not limited to,chloramphenicol acetyl transferase (CAT), β-galactosidase (encoded bythe lacZ gene), luciferase (encoded by the luc gene), alkalinephosphatase (AP), green fluorescent protein (GFP), and horseradishperoxidase (HRP). Such vectors and assays are readily available, from,inter alia, commercial sources. Plasmids thus constructed aretransfected into a suitable host cell to test for expression of thereporter gene as controlled by the putative TRE using transfectionmethods known in the art, such as calcium phosphate precipitation,electroporation, liposomes, DEAE dextran-mediated transfer, particlebombardment or direct injection. TRE activity is measured by detectionand/or quantitation of reporter gene-derived mRNA and/or protein.Reporter protein product can be detected directly (e.g.,immunochemically) or through its enzymatic activity, if any, using anappropriate substrate. Generally, to determine cell specific activity ofa TRE, a TRE-reporter gene construct is introduced into a variety ofcell types. The amount of TRE activity is determined in each cell typeand compared to that of a reporter gene construct lacking the TRE. A TREis determined to be cell-specific if it is preferentially functional inone cell type, compared to a different type of cell.

A hepatocellular carcinoma specific TRE comprises a mammalianhepatocellular carcinoma-specific enhancer and/or promoter. Preferredmetastatic cancer-specific enhancers and/or promoters for use inpracticing the invention are of human, rat or mouse origin, includingpromoter and enhancer elements and transcription factor bindingsequences from the 5′ CRG-L2 sequence set forth in SEQ ID NO:1. In oneembodiment, the TRE comprises SEQ ID NO:1, residues 119-803.Transcriptional regulatory sequences of interest known to conferspecificity include the promoter and enhancer elements from CRG-L2 asdescribed herein. This region of DNA contains the native promoterelements that direct expression of the linked gene. Usually a promoterregion will have at least about 100 nt of sequence located 5′ to thegene and further comprising a TATA box and CAAT box motif sequence.

The sequence of this 5′ region, and further 5′ upstream sequences may beutilized to direct gene expression, including enhancer binding sites,that provide for expression in tissues where CRG-L2 is expressed, e.g.hepatocellular carcinoma cells. The tissue specific expression is usefulfor determining the pattern of expression, and for providing promotersthat mimic the native pattern of expression. Sequence alterations,including substitutions, deletions and additions, may be introduced intothe promoter region to determine the effect of altering expression inexperimentally defined systems. Methods for the identification ofspecific DNA motifs involved in the binding of transcriptional factorsare known in the art, e.g. sequence similarity to known binding motifs,gel retardation studies, etc.

The regulatory sequences may be used to identify cis acting sequencesrequired for transcriptional or translational regulation of CRG-L2expression, especially in different stages of metastasis, and toidentify cis acting sequences and trans acting factors that regulate ormediate expression. Such transcription or translational control regionsmay be operably linked to a gene of interest in order to promoteexpression of a protein of interest in cultured cells, or in embryonic,fetal or adult tissues, and for gene therapy.

A hepatocellular carcinoma-specific TRE can also comprise multimers. Forexample, a hepatocellular carcinoma-specific TRE can comprise a tandemseries of at least two, at least three, at least four, or at least fivepromoter fragments. Alternatively, a hepatocellular carcinoma-specificTRE could have one or more promoter regions along with one or moreenhancer regions. These multimers may also contain heterologous promoterand/or enhancer sequences and/or transcription factor binding sites.

The promoter enhancer and/or transcription factor binding sitecomponents of a hepatocellular carcinoma-specific TRE may be in anyorientation and/or distance from the coding sequence of interest, aslong as the desired target cell-specific transcriptional activity isobtained. Transcriptional activation can be measured in a number of waysknown in the art, but is generally measured by detection and/orquantitation of mRNA or the protein product of the coding sequence undercontrol of (i.e., operably linked to) the hepatocellularcarcinoma-specific TRE. As discussed herein, a hepatocellularcarcinoma-specific TRE can be of varying lengths, and of varyingsequence composition.

As is known in the art, the activity of TREs can be inducible. InducibleTREs generally exhibit low activity in the absence of inducer, and areup-regulated in the presence of inducer. Inducers include, for example,nucleic acids, polypeptides, small molecules, organic compounds and/orenvironmental conditions such as temperature, pressure or hypoxia.Inducible TREs may be preferred when expression is desired only atcertain times or at certain locations, or when it is desirable totitrate the level of expression using an inducing agent.

A TRE for use in the present vectors may or may not comprise a silencer.The presence of a silencer (i.e., a negative regulatory element known inthe art) can assist in shutting off transcription (and thus replication)in non-target cells. Thus, the presence of a silencer can conferenhanced cell-specific vector replication by more effectively preventingreplication in non-target cells. Alternatively, the lack of a silencermay stimulate replication in target cells, thus conferring enhancedtarget cell-specificity.

A “functionally-preserved variant” of a hepatocellularcarcinoma-specific TRE differs, usually in sequence, but still retainsthe biological activity, e.g., target cell-specific transcriptionactivity of the corresponding native or parent hepatocellularcarcinoma-specific TRE, although the degree of activation may bealtered. The difference in sequence may arise from, for example, singlebase mutation(s), addition(s), deletion(s), and/or modification(s) ofthe bases. The difference can also arise from changes in the sugar(s),and/or linkage(s) between the bases of a hepatocellularcarcinoma-specific TRE. For example, certain point mutations withinsequences of TREs have been shown to decrease transcription factorbinding and stimulation of transcription (see Blackwood, et al. (1998)Science 281:60-63, and Smith et al. (1997) J. Biol. Chem.272:27493-27496). One of skill in the art would recognize that somealterations of bases in and around transcription factor binding sitesare more likely to negatively affect stimulation of transcription andcell-specificity, while alterations in bases that are not involved intranscription factor binding are not as likely to have such effects.Certain mutations are also capable of increasing TRE activity. Testingof the effects of altering bases may be performed in vitro or in vivo byany method known in the art, such as mobility shift assays, ortransfecting vectors containing these alterations in TRE functional andTRE non-functional cells. Additionally, one of skill in the art wouldrecognize that point mutations and deletions can be made to a TREsequence without altering the ability of the sequence to regulatetranscription. It will be appreciated that typically a“functionally-preserved variant” of a hepatocellular carcinoma-specificTRE will hybridize to the parent sequence under conditions of highstringency. Exemplary high stringency conditions include hybridizationat about 65° C. in about 5×SSPE and washing at about 65° C. in about0.1×SSPE (where 1×SSPE=0.15 sodium chloride, 0.010 M sodium phosphate,and 0.001 M disodium EDTA). Further examples of high stringencyconditions are provided in: Maniatis, et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2d Edition (1989); and Ausubel, F. M., et al., Eds.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc.,Copyright (c)1987, 1988, 1989, 1990 by Current Protocols, both of whichare hereby incorporated by reference.

In some instances, a “functionally-preserved variant” of ahepatocellular carcinoma-specific TRE is a fragment of a native orparent hepatocellular carcinoma-specific TRE. The term “fragment,” whenreferring to a hepatocellular carcinoma-specific TRE, refers to asequence that is the same as part of, but not all of, the nucleic acidsequence of a native or parental hepatocellular carcinoma-specific TRE.Such a fragment either exhibits essentially the same biological functionor activity as the native or parental hepatocellular carcinoma-specificTRE; for example, a fragment which retains the target cell-specifictranscription activity of the corresponding native or parenthepatocellular carcinoma-specific TRE, although the degree of activationmay be altered.

As used herein, an “internal ribosome entry site” or “IRES” refers to anelement that promotes direct internal ribosome entry to the initiationcodon, such as ATG, of a cistron (a protein encoding region), therebyleading to the cap-independent translation of the gene (Jackson et al.(1990) Trends Biochem Sci 15(12):477-83) and Jackson et al. (1995) RNA1(10):985-1000). The present invention encompasses the use of any IRESelement that is able to direct internal ribosome entry to the initiationcodon of a cistron. “Under translational control of an IRES” as usedherein means that translation is associated with the IRES and proceedsin a cap-independent manner. Examples of “IRES” known in the artinclude, but are not limited to IRES obtainable from picornavirus(Jackson et al., 1990, Trends Biochem Sci 15(12):477-483); and IRESobtainable from viral or cellular mRNA sources, such as for example,immunogloublin heavy-chain binding protein (BiP), the vascularendothelial growth factor (VEGF) (Huez et al. (1998) Mol Cell. Biol.18(11):6178-6190), the fibroblast growth factor 2, and insulin-likegrowth factor, the translational initiation factor eIF4G, yeasttranscription factors TFIID and HAP4. IRES have also been reported indifferent viruses such as cardiovirus, rhinovirus, aphthovirus, HCV,Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus(MoMLV). As used herein, the term “IRES” encompasses functionalvariations of IRES sequences as long as the variation is able to promotedirect internal ribosome entry to the initiation codon of a cistron. Inpreferred embodiments, the IRES is mammalian. In other embodiments, theIRES is viral or protozoan. In one illustrative embodiment disclosedherein, the IRES is obtainable from encephelomycarditis virus (ECMV)(commercially available from Novogen, Duke et al. (1992) J. Virol66(3):1602-1609). In another illustrative embodiment disclosed herein,the IRES is from VEGF. In some embodiments, an adenovirus vectorcomprising an IRES and a target cell-specific TRE exhibits greaterspecificity for the target cell than an adenovirus vector comprising atarget cell-specific TRE and lacking an IRES.

In some embodiments, specificity is conferred by preferentialtranscription and/or translation of the first and second genes due tothe presence of a target cell specific TRE. In other embodiments,specificity is conferred by preferential replication of the adenovirusvectors in target cells due to the target cell-specific TRE drivingtranscription of a gene essential for replication.

An “E3 region” (used interchangeably with “E3”) is a term wellunderstood in the art and means the region of the adenoviral genome thatencodes the E3 gene products. Generally, the E3 region is locatedbetween about nucleotides 28583 and 30470 of the adenoviral genome. TheE3 region has been described in various publications, including, forexample, Wold et al. (1995) Curr. Topics Microbiol. Immunol.199:237-274. A “portion” of the E3 region means less than the entire E3region, and as such includes polynucleotide deletions as well aspolynucleotides encoding one or more polypeptide products of the E3region.

A “multicistronic transcript” refers to an mRNA molecule that containsmore than one protein coding region, or cistron. A mRNA comprising twocoding regions is denoted a “bicistronic transcript.” The “5′-proximal”coding region or cistron is the coding region whose translationinitiation codon (usually AUG) is closest to the 5′-end of amulticistronic mRNA molecule. A “5′-distal” coding region or cistron isone whose translation initiation codon (usually AUG) is not the closestinitiation codon to the 5′ end of the mRNA. The terms “5′-distal” and“downstream” are used synonymously to refer to coding regions that arenot adjacent to the 5′ end of a mRNA molecule.

As used herein, “co-transcribed” means that two (or more) coding regionsof polynucleotides are under transcriptional control of a singletranscriptional control element.

As used herein, the term “vector” refers to a polynucleotide constructdesigned for transduction/transfection of one or more cell types.Vectors may be, for example, “cloning vectors” which are designed forisolation, propagation and replication of inserted nucleotides,“expression vectors” which are designed for expression of a nucleotidesequence in a host cell, or a “viral vector” which is designed to resultin the production of a recombinant virus or virus-like particle, or“shuttle vectors”, which comprise the attributes of more than one typeof vector. Any vector for use in gene introduction can basically be usedas a “vector” into which the DNA having promoter activity is to beintroduced. Particularly in gene therapy, viral vectors, such asretrovirus vectors, adenovirus vectors, or adeno associated virusvectors, and non-viral vectors such as liposomes should be used. Plasmidvectors may also find use. Any cell is included in the “cell carryingthe expression vector” of the present invention.

The term “plasmid” as used herein refers to a DNA molecule that iscapable of autonomous replication within a host cell, eitherextrachromosomally or as part of the host cell chromosome(s). Thestarting plasmids herein are commercially available, are publiclyavailable on an unrestricted basis, or can be constructed from suchavailable plasmids as disclosed herein and/or in accordance withpublished procedures. In certain instances, as will be apparent to theordinarily skilled artisan, other plasmids known in the art may be usedinterchangeable with plasmids described herein.

Methods that are well known to those skilled in the art can be used toconstruct expression vectors containing coding sequences and appropriatetranscriptional and translational control signals, including a livercancer specific control signal, for specific expression of an exogenousgene introduced into a cell. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Alternatively, RNA capable of encoding geneproduct sequences may be chemically synthesized using, for example,synthesizers. See, for example, the techniques described in“Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., IRL Press, Oxford.

In mammalian host cells, a number of viral-based expression systems maybe utilized, including retroviral vectors (e.g. derived from MoMLV,MSCV, SFFV, MPSV, SNV etc), lentiviral vectors (e.g. derived from HIV-1,HIV-2, SIV, BIV, FIV etc.), adeno-associated virus (MV) vectors,adenoviral vectors (e.g. derived from Ad5 virus), SV40-based vectors,Herpes Simplex Virus (HSV)-based vectors etc. In cases where anadenovirus is used as an expression vector, the coding sequence ofinterest may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing the gene product ininfected hosts (see Logan & Shenk, 1984, Proc. Nati. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted gene product coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Standard systems for generating adenoviral vectors for expression oninserted sequences are available from commercial sources, for examplethe Adeno-™ expression system from Clontech (Clontechniques (January2000) p. 10-12).

In cases where an entire gene, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional translational control signals may be needed. However, incases where only a portion of the gene coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, must be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:516-544).

An “adenovirus vector” or “adenoviral vector” (used interchangeably) ofthe invention is a polynucleotide construct, which is replicationcompetent, exhibits preferential replication in primary livercancercells and contains a tissue-specific transcriptional regulatorysequence linked to an adenoviral gene. In some embodiments, anadenoviral vector of the invention includes a therapeutic gene sequence,e.g., a cytokine gene sequence. Exemplary adenoviral vectors of theinvention include, but are not limited to, DNA, DNA encapsulated in anadenovirus coat, adenoviral DNA packaged in another viral or viral-likeform (such as herpes simplex, and AAV), adenoviral DNA encapsulated inliposomes, adenoviral DNA complexed with polylysine, adenoviral DNAcomplexed with synthetic polycationic molecules, conjugated withtransferrin, or complexed with compounds such as PEG to immunologically“mask” the antigenicity and/or increase half-life, or conjugated to anonviral protein.

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include a single-,double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid,or a polymer comprising purine and pyrimidine bases, or other natural,chemically, biochemically modified, non-natural or derivatizednucleotide bases. Preferably, an adenoviral polynucleotide is DNA. Asused herein, “DNA” includes not only bases A, T, C, and G, but alsoincludes any of their analogs or modified forms of these bases, such asmethylated nucleotides, internucleotide modifications such as unchargedlinkages and thioates, use of sugar analogs, and modified and/oralternative backbone structures, such as polyamides. For purposes ofthis invention, adenovirus vectors are replication-competent in a targetcell.

The following are non-limiting examples of polynucleotides: a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications included in thisdefinition are caps, substitution of one or more of the naturallyoccurring nucleotides with an analog, and introduction of means forattaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support. Preferably, thepolynucleotide is DNA. As used herein, “DNA” includes not only bases A,T, C, and G, but also includes any of their analogs or modified forms ofthese bases, such as methylated nucleotides, internucleotidemodifications such as uncharged linkages and thioates, use of sugaranalogs, and modified and/or alternative backbone structures, such aspolyamides.

A polynucleotide or polynucleotide region has a certain percentage (forexample, 80%, 85%, 90%, or 95%) of “sequence identity” to anothersequence means that, when aligned, that percentage of bases are the samein comparing the two sequences. This alignment and the percent homologyor sequence identity can be determined using software programs known inthe art, for example those described in Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987) Supplement 30, section7.7.18. A preferred alignment program is ALIGN Plus (Scientific andEducational Software, Pennsylvania), preferably using defaultparameters, which are as follows: mismatch=2; open gap=0; extend gap=2.

In the context of adenovirus, a “heterologous polynucleotide” or“heterologous gene” or “transgene” is any polynucleotide or gene that isnot present in wild-type adenovirus. Preferably, the transgene will alsonot be expressed or present in the target cell prior to introduction bythe adenovirus vector. Examples of preferred transgenes for inclusion inthe adenovirus vectors of the invention, are provided below.

In the context of adenovirus, a “heterologous” promoter or enhancer isone which is not associated with or derived from an adenovirus gene.

In the context of a target cell-specific TRE, a “heterologous” promoteror enhancer is one which is derived from a gene other than the gene fromwhich a particular target cell-specific TRE is derived.

In the context of adenovirus, an “endogenous” promoter, enhancer, or TREis native to or derived from adenovirus.

“Replication” and “propagation” are used interchangeably and refer tothe ability of an adenovirus vector of the invention to reproduce orproliferate. These terms are well understood in the art. For purposes ofthis invention, replication involves production of adenovirus proteinsand is generally directed to reproduction of adenovirus. Replication canbe measured using assays standard in the art and described herein, suchas a virus yield assay, burst assay or plaque assay. “Replication” and“propagation” include any activity directly or indirectly involved inthe process of virus manufacture, including, but not limited to, viralgene expression; production of viral proteins, nucleic acids or othercomponents; packaging of viral components into complete viruses; andcell lysis.

“Preferential replication” and “selective replication” may be usedinterchangeably and mean that an adenovirus replicates more in a targetcell than in a non-target cell. Preferably, the adenovirus replicates ata significantly higher rate in target cells than non target cells;preferably, at least about 5-fold higher, more preferably, at leastabout 10-fold higher, still more preferably at least about 50-foldhigher, even more preferably at least about 100-fold higher, still morepreferably at least about 400- to 500-fold higher, still more preferablyat least about 1000-fold higher, most preferably at least about 1×10⁶higher. Most preferably, the adenovirus replicates only in the targetcells (that is, does not replicate at all or replicates at a very lowlevel in non-target cells).

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals, sportanimals, rodents, primates, and pets. A “host cell” includes anindividual cell or cell culture which can be or has been a recipient ofan adenoviral vector(s) of this invention. Host cells include progeny ofa single host cell, and the progeny may not necessarily be completelyidentical (in morphology or in total DNA complement) to the originalparent cell due to natural, accidental, or deliberate mutation and/orchange. A host cell includes cells transfected or infected in vivo or invitro with an adenoviral vector of this invention.

As used herein, “cytotoxicity” is a term well understood in the art andrefers to a state in which a cell's usual biochemical or biologicalactivities are compromised (i.e., inhibited). These activities include,but are not limited to, metabolism; cellular replication; DNAreplication; transcription; translation; uptake of molecules.“Cytotoxicity” includes cell death and/or cytolysis. Assays are known inthe art which indicate cytotoxicity, such as dye exclusion, ³H-thymidineuptake, and plaque assays.

The term “selective cytotoxicity”, as used herein, refers to thecytotoxicity conferred by an adenovirus vector of the invention on acell which allows or induces a target cell-specific TRE to function(referred to herein as a “target cell”) when compared to thecytotoxicity conferred by an adenoviral vector of the present inventionon a cell which does not allow a target cell-specific TRE to function (a“non-target cell”). Such cytotoxicity may be measured, for example, byplaque assays, by reduction or stabilization in size of a tumorcomprising target cells, or the reduction or stabilization of serumlevels of a marker characteristic of the tumor cells, or atissue-specific marker, e.g., a cancer marker.

Adenoviral Vectors

The adenoviral vectors used in the methods described herein arereplication-competent liver cancer-specific adenoviral vectorscomprising an adenovirus gene, preferably a gene essential forreplication under transcriptional control of a hepatocellularcarcinoma-specific TRE. The vector may or may not include an E3 region.In other embodiments, an adenovirus vector is a replication competentliver cancer-specific vector comprising E1B, wherein E1B has a deletionof part or all of the 19-kDa region. In some embodiments the adenoviralgene essential for replication is an early gene, preferably E1A or E1Bor both. In some embodiments, the adenovirus vector comprisesco-transcribed first and second genes under transcriptional control of aheterologous, liver cancer-specific transcriptional regulatory element(TRE), wherein the second gene is under translational control of aninternal ribosome entry site (IRES). The adenovirus vector may furthercomprise E3.

The E1B 19-kDa region refers to the genomic region of the adenovirus E1Bgene encoding the E1B 19-kDa product. According to wild-type Ad5 , theE1B 19-kDa region is a 261 bp region located between nucleotide 1714 andnucleotide 2244. The E1B 19-kDa region has been described in, forexample, Rao et al., Proc. Natl. Acad. Sci. USA, 89:7742-7746. Thepresent invention encompasses deletion of part or all of the E1B 19-kDaregion as well as embodiments wherein the E1B 19-kDa region is mutated,as long as the deletion or mutation lessens or eliminates the inhibitionof apoptosis associated with E1B-19kDa.

The adenovirus vectors used in this invention replicate preferentiallyin hepatocellular carcinoma cells, which replication preference isindicated by comparing the level of replication (i.e., titer) inhepatocellular carcinoma cells to the level of replication innon-hepatpcellular carcinoma cells, normal or control cells. Comparisonof the adenovirus titer of a hepatocellular carcinoma cell to the titerof a TRE inactive cell type provides a key indication that the overallreplication preference is enhanced due to the replication in targetcells as well as depressed replication in non-target cells. This isespecially useful in the metastatic cancer context, in which targetedcell killing is desirable. Runaway infection is prevented due to thecell-specific requirements for viral replication. Without wishing to bebound by any particular theory, production of adenovirus proteins canserve to activate and/or stimulate the immune system, either generallyor specifically toward target cells producing adenoviral proteins whichcan be an important consideration in the cancer context, whereindividuals are often moderately to severely immunocompromised.

In one aspect of the present invention, the adenovirus vectors comprisean intergenic IRES element(s) which links the translation of two or moregenes, thereby removing any potential for homologous recombination basedon the presence of identical TREs in the vector. Adenovirus vectorscomprising an IRES are stable and in some embodiments provide betterspecificity than vectors not containing an IRES. Another advantage of anadenovirus vector comprising an intergenic IRES is that the use of anIRES rather than a second TRE may provide additional space in the vectorfor an additional gene(s) such as a therapeutic gene. Accordingly, inone aspect of the invention, the viral vectors disclosed hereintypically comprise at least one IRES within a multicistronic transcript,wherein production of the multicistronic transcript is regulated by aheterologous, target cell-specific TRE. For adenovirus vectorscomprising a second gene under control of an IRES, it is preferred thatthe endogenous promoter of the gene under translational control of anIRES be deleted so that the endogenous promoter does not interfere withtranscription of the second gene. It is preferred that the second genebe in frame with the IRES if the IRES contains an initiation codon. Ifan initiation codon, such as ATG, is present in the IRES, it ispreferred that the initiation codon of the second gene is removed andthat the IRES and the second gene are in frame. Alternatively, if theIRES does not contain an initiation codon or if the initiation codon isremoved from the IRES, the initiation codon of the second gene is used.In one embodiment, the adenovirus vectors comprise the adenovirusessential genes, E1A and E1B genes, under the transcriptional control ofa heterologous hepatocellular carcinoma-specific TRE, and an IRESintroduced between E1A and E1B. Thus, both E1A and E1B are under commontranscriptional control, and translation of E1B coding region isobtained by virtue of the presence of the IRES. In one embodiment, E1Ahas its endogenous promoter deleted. In another embodiment, E1A has anendogenous enhancer deleted and in yet an additional embodiment, E1A hasits endogenous promoter deleted and E1A enhancer deleted. In anotherembodiment, E1B has its endogenous promoter deleted. In yet furtherembodiments, E1B has a deletion of part or all of the 19-kDa region ofE1B.

An adenovirus vector may further include an additional heterologous TREwhich may or may not be operably linked to the same gene(s) as thetarget cell-specific TRE. For example a TRE (such as a celltype-specific or cell status-specific TRE) may be juxtaposed to a secondtype of target-cell-specific TRE. “Juxtaposed” means a targetcell-specific TRE and a second TRE transcriptionally control the samegene. For these embodiments, the target cell-specific TRE and the secondTRE may be in any of a number of configurations, including, but notlimited to, (a) next to each other (i.e., abutting); (b) both 5′ to thegene that is transcriptionally controlled (i.e., may have interveningsequences between them); (c) one TRE 5′ and the other TRE 3′ to thegene.

To provide cytotoxicity to target cells, one or more transgenes having acytotoxic effect may be present in the vector. Additionally, oralternatively, an adenovirus gene that contributes to cytotoxicityand/or cell death, such as the adenovirus death protein (ADP) gene, canbe included in the vector, optionally under the selectivetranscriptional control of a heterologous TRE and optionally under thetranslational control of an IRES.

In some embodiments, an adenovirus vector of the invention comprises atransgene, which may confer a therapeutic effect, such as enhancingcytotoxicity so as to eliminate unwanted target cells. The transgene maybe under the transcriptional control of a hepatocellular carcinoma TRE,e.g. a CRG-L2 TRE, which may comprise a CRG-L2 promoter. The transgenemay be regulated independently of the adenovirus gene regulation, i.e.having separate promoters, which may the same or different, or may becoordinately regulated, i.e. having a single promoter in conjunctionwith an IRES.

In this way, various genetic capabilities may be introduced into targetcells, particularly cancer cells. For example, in certain instances, itmay be desirable to enhance the degree and/or rate of cytotoxicactivity, due to, for example, the relatively refractory nature orparticular aggressiveness of the cancerous target cell. This could beaccomplished by coupling the target cell-specific cytotoxic activitywith cell-specific expression of, for example, HSV-tk which and/orcytosine deaminase (cd). Cancer cells can be induced to be conditionallysensitive to the antiviral drug ganciclovir after transduction withHSV-tk. Ganciclovir is converted by HSV-tk into its triphosphate form bycellular enzymes and incorporated into the DNA of replicating mammaliancells leading to inhibition of DNA replication and cell death. Cytosinedeaminase renders cells capable of metabolizing 5-fluorocytosine (5-FC)to the chemotherapeutic agent 5-flurouracil (5-FU).

Other desirable transgenes that may be introduced via an adenovirusvector(s) include genes encoding cytokines such as interferons andinterleukins; genes encoding lymphokines; genes coding for membranereceptors such as the receptors recognized by pathogenic organisms(viruses, bacteria or parasites), preferably by the HIV virus (humanimmunodeficiency virus); genes coding for coagulation factors such asfactor VIII and factor IX; genes coding for dystrophins; genes codingfor insulin; genes coding for proteins participating directly orindirectly in cellular ion channels, such as the CFTR (cystic fibrosistransmembrane conductance regulator) protein; genes coding for antisenseRNAs, or proteins capable of inhibiting the activity of a proteinproduced by a pathogenic gene which is present in the genome of apathogenic organism, or proteins (or genes encoding them) capable ofinhibiting the activity of a cellular gene whose expression isderegulated, for example an oncogene; genes coding for a proteininhibiting an enzyme activity, such as α1-antitrypsin or a viralprotease inhibitor, for example; genes coding for variants of pathogenicproteins which have been mutated so as to impair their biologicalfunction, such as, for example, trans-dominant variants of the tatprotein of the HIV virus which are capable of competing with the naturalprotein for binding to the target sequence, thereby preventing theactivation of HIV; genes coding for antigenic epitopes in order toincrease the host cell's immunity; genes coding for majorhistocompatibility complex classes I and II proteins, as well as thegenes coding for the proteins which are inducers of these genes; genescoding for antibodies; genes coding for immunotoxins; genes encodingtoxins; genes encoding growth factors or growth hormones; genes encodingcell receptors and their ligands; genes encoding tumor suppressors;genes coding for cellular enzymes or those produced by pathogenicorganisms; and suicide genes.

Cytotoxic genes of interest include the A chains of diphtheria toxin,ricin or abrin (Palmiter et al. (1987) Cell 50: 435; Maxwell et al.(1987) Mol. Cell. Biol 7: 1576; Behringer et al. (1988) Genes Dev. 2:453; Messing et al. (1992) Neuron 8: 507; Piatak et al. (1988) J. Biol.Chem. 263: 4937; Lamb et al. (1985) Eur. J. Biochem. 148: 265; Frankelet al. (1989) MoL Cell. Biol. 9: 415), genes encoding a factor capableof initiating apoptosis, sequences encoding antisense transcripts orribozymes, which among other capabilities may be directed to mRNAsencoding proteins essential for proliferation, such as structuralproteins, or transcription factors; viral or other pathogenic proteins,where the pathogen proliferates intracellularly; genes that encode anengineered cytoplasmic variant of a nuclease (e.g. RNase A) or protease(e.g. pepsin, papain, proteinase K, carboxypeptidase, etc.), or encodethe Fas gene, and the like.

The HSV-1 TK suicide gene may be mentioned as an example. This viral TKenzyme displays markedly greater affinity compared to the cellular TKenzyme for certain nucleoside analogues (such as acyclovir organcyclovir). It converts them to monophosphorylated molecules, whichcan themselves be converted by cellular enzymes to nucleotideprecursors, which are toxic. These nucleotide analogues can beincorporated into replicating DNA molecules, hence incorporation occurschiefly in the DNA of dividing cells. This incorporation can result inspecific destruction of dividing cells such as cancer cells.

Although any gene or coding sequence of therapeutic relevance can beused in the practice of the invention, certain genes, or fragmentsthereof, are particularly suitable. For example, genes encodingimmunogenic polypeptides, toxins, immunotoxins and cytokines are usefulin the practice of the invention. Cytokine genes of use in the inventioninclude, but are not limited to, those encoding α, β or γ interferon(IFN), interleukins (IL) such as IL-2, IL-6, IL-10 or IL-12, tumornecrosis factor (TNF), livery stimulating factors such as GM-CSF, C-CSF,M-CSF, and other cytokines as are known to those of skill in the art.Additional genes include those encoding cell or nuclear receptors andtheir ligands (e.g., fas ligand), coagulation factors (for example,FVIII, FIX), growth hormones, growth factors such as fibroblast growthfactors (FGF), vascular endothelial growth factors (VEGF), nerve growthfactors (NGF), epidermal growth factors (EGF), platelet-derived growthfactors (PDGF) and other growth factors as are known to those of skillin the art. Genes suitable for use in the practice of the invention canencode enzymes (such as, for example, urease, renin, thrombin,metalloproteases, nitric oxide synthase, superoxide dismutase, catalaseand others known to those of skill in the art), enzyme inhibitors (suchas, for example, α1-antitrypsin, antithrombin III, cellular or viralprotease inhibitors, plasminogen activator inhibitor-1, tissue inhibitorof metalloproteases, etc.), the cystic fibrosis transmembraneconductance regulator (CFTR) protein, insulin, dystrophin, or a MajorHistocompatibility Complex (MHC) antigen of class I or II. Also usefulare genes encoding polypeptides that can modulate/regulate expression ofcorresponding genes, polypeptides capable of inhibiting a bacterial,parasitic or viral infection or its development (for example, antigenicpolypeptides, antigenic epitopes, and transdominant protein variantsinhibiting the action of a native protein by competition), apoptosisinducers or inhibitors (for example, Bax, Bc12, Bc1X and others known tothose of skill in the art), cytostatic agents (e.g., p21, p16, Rb,etc.), apolipoproteins (e.g., ApoAI, ApoAIV, ApoE, etc.), angiogenesisinhibitors (e.g., angiostatin, endostatin, etc.), oxygen radicalscavengers, polypeptides having an anti-tumor effect, antibodies,toxins, immunotoxins, markers (e.g., β-galactosidase, luciferase, etc.)or any other genes of interest that are recognized in the art as beinguseful for treatment or prevention of a clinical condition.

Suitable genes of interest to delay or inhibit tumor or cancerprogression, include but are not limited to those encoding an antisenseRNA, a ribozyme, a cytotoxic product such as thymidine kinase of herpessimplex virus type 1 (HSV-1 TK), ricin, a bacterial toxin, the productsof the yeast genes FCY1 and/or FUR1 having CDase (cytosine deaminase)and UPRTase (uracil phosphoribosyl transferase) activities respectively,an antibody, a polypeptide inhibiting cellular division or signaltransduction, a tumor suppressor gene (such as, for example, p53, Rb,p73), a polypeptide which activates the host immune system, atumor-associated antigen (e.g., MUC-1, BRCA-1, an HPV early or lateantigen such as E6, E7, L1, L2, etc), optionally in combination with acytokine gene.

Functionally preserved variants of TREs can be used in the vectorsdisclosed herein. Variant TREs retain function in the target cell butneed not exhibit maximal function. In fact, maximal transcriptionalactivation activity of a TRE may not always be necessary to achieve adesired result, and the level of induction afforded by a fragment of aTRE may be sufficient for certain applications. For example, if used fortreatment or palliation of a disease state, less-than-maximalresponsiveness may be sufficient if, for example, the target cells arenot especially virulent and/or the extent of disease is relativelyconfined.

As discussed herein, a TRE can be of varying lengths, and of varyingsequence composition. The size of a heterologous TRE will be determinedin part by the capacity of the viral vector, which in turn depends uponthe contemplated form of the vector (see infra). Generally minimal sizesare preferred for TREs, as this provides potential room for insertion ofother sequences which may be desirable, such as transgenes, and/oradditional regulatory sequences. In a preferred embodiment, such anadditional regulatory sequence is an IRES. However, if no additionalsequences are contemplated, or if, for example, an adenoviral vectorwill be maintained and delivered free of any viral packagingconstraints, larger TRE sequences can be used as long as the resultantadenoviral vector remains replication-competent.

To minimize non-specific replication, endogenous adenovirus TREs arepreferably removed from the vector. Besides facilitating targetcell-specific replication, removal of endogenous TREs also providesgreater insert capacity in a vector, which may be of special concern ifan adenoviral vector is to be packaged within a virus particle. Evenmore importantly, deletion of endogenous TREs prevents the possibilityof a recombination event whereby a heterologous TRE is deleted and theendogenous TRE assumes transcriptional control of its respectiveadenovirus coding sequences. However, endogenous TREs can be maintainedin the adenovirus vector(s), provided that sufficient cell-specificreplication preference is preserved. These embodiments are constructedby inserting heterologous TREs between an endogenous TRE and areplication gene coding segment. Requisite liver cancer-specificreplication preference is determined by conducting assays that comparereplication of the adenovirus vector in a cell which allows function ofthe heterologous TREs with replication in a cell which does not.

The adenovirus vectors of this invention can be prepared usingrecombinant techniques that are standard in the art. Generally, a livercancer-specific TRE is inserted 5′ to the adenoviral gene of interest,preferably an adenoviral replication gene, more preferably one or moreearly replication genes (although late gene(s) can be used). Ahepatocellular carcinoma-specific TRE can be prepared usingoligonucleotide synthesis (if the sequence is known) or recombinantmethods (such as PCR and/or restriction enzymes). Convenient restrictionsites, either in the natural adeno-DNA sequence or introduced by methodssuch as PCR or site-directed mutagenesis, provide an insertion site fora hepatocellular carcinoma-specific TRE. Accordingly, convenientrestriction sites for annealing (i.e., inserting) a hepatocellularcarcinoma-specific TRE can be engineered onto the 5′ and 3′ ends of aUP-TRE using standard recombinant methods, such as PCR.

Adenoviral vectors containing all replication-essential elements, withthe desired elements (e.g., E1A) under control of a livercancer-specific TRE, are conveniently prepared by homologousrecombination or in vitro ligation of two plasmids, one providing theleft-hand portion of adenovirus and the other plasmid providing theright-hand region, one or more of which contains at least one adenovirusgene under control of a hepatocellular carcinoma-specific TRE. Ifhomologous recombination is used, the two plasmids should share at leastabout 500 bp of sequence overlap. Each plasmid, as desired, may beindependently manipulated, followed by cotransfection in a competenthost, providing complementing genes as appropriate, or the appropriatetranscription factors for initiation of transcription from ahepatocellular carcinoma-specific TRE for propagation of the adenovirus.Plasmids are generally introduced into a suitable host cell such as 293cells using appropriate means of transduction, such as cationicliposomes. Alternatively, in vitro ligation of the right and left-handportions of the adenovirus genome can also be used to constructrecombinant adenovirus derivative containing all thereplication-essential portions of adenovirus genome. Berkner et al.(1983) Nucleic Acid Research 11: 6003-6020; Bridge et al. (1989) J.Virol 63: 631-638.

For convenience, plasmids are available that provide the necessaryportions of adenovirus. Plasmid pXC.1 (McKinnon (1982) Gene 19:3342)contains the wild-type left-hand end of Ad5. pBHG10 (Bett et al. (1994);Microbix Biosystems Inc., Toronto) provides the right-hand end of Ad5,with a deletion in E3. The deletion in E3 provides room in the virus toinsert a 3 kb TRE without deleting the endogenous enhancer/promoter. Thegene for E3 is located on the opposite strand from E4 (r-strand). pBHG11provides an even larger E3 deletion (an additional 0.3 kb is deleted).Bett et al. (1994). Alternatively, the use of pBHGE3 (MicrobixBiosystems, Inc.) provides the right hand end of Ad5, with a full-lengthof E3.

For manipulation of the early genes, the transcription start site of Ad5E1A is at 498 and the ATG start site of the E1A coding segment is at 560in the virus genome. This region can be used for insertion of a livercancer-specific TRE. A restriction site may be introduced by employingpolymerase chain reaction (PCR), where the primer that is employed maybe limited to the Ad5 genome, or may involve a portion of the plasmidcarrying the Ad5 genomic DNA. For example, where pBR322 is used, theprimers may use the EcoRI site in the pBR322 backbone and the XbaI siteat nt 1339 of Ad5. By carrying out the PCR in two steps, whereoverlapping primers at the center of the region introduce a nucleotidesequence change resulting in a unique restriction site, one can providefor insertion of a hepatocellular carcinoma-specific TRE at that site.

A similar strategy may also be used for insertion of a livercancer-specific TRE element to regulate E1B. The E1B promoter of Ad5consists of a single high-affinity recognition site for SpI and a TATAbox. This region extends from Ad5 nt 1636 to 1701. By insertion of a TREin this region, one can provide for cell-specific transcription of theE1B gene. By employing the left-hand region modified with thecell-specific response element regulating E1A, as the template forintroducing a liver cancer-specific TRE to regulate E1B, the resultingadenovirus vector will be dependent upon the cell-specific transcriptionfactors for expression of both E1A and E1B. In some embodiments, part orall of the 19-kDa region of E1B is deleted.

Similarly, a liver cancer-specific TRE can be inserted upstream of theE2 gene to make its expression cell-specific. The E2 early promoter,mapping in Ad5 from 27050-27150, consists of a major and a minortranscription initiation site, the latter accounting for about 5% of theE2 transcripts, two non-canonical TATA boxes, two E2F transcriptionfactor binding sites and an ATF transcription factor binding site (for adetailed review of the E2 promoter architecture see Swaminathan et al.,Curr. Topics in Micro. and Immunol (1995) 199(part 3):177-194.

The E2 late promoter overlaps with the coding sequences of a geneencoded by the counterstrand and is therefore not amenable for geneticmanipulation. However, the E2 early promoter overlaps only for a fewbase pairs with sequences coding for a 33 kD protein on thecounterstrand. Notably, the SpeI restriction site (Ad5 position 27082)is part of the stop codon for the above mentioned 33 kD protein andconveniently separates the major E2 early transcription initiation siteand TATA-binding protein site from the upstream transcription factorbinding sites E2F and ATF. Therefore, insertion of a hepatocellularcarcinoma-specific TRE having SpeI ends into the SpeI site in the1-strand would disrupt the endogenous E2 early promoter of Ad5 andshould allow cell-restricted expression of E2 transcripts.

For E4, one must use the right hand portion of the adenovirus genome.The E4 transcription start site is predominantly at about nt 35605, theTATA box at about nt 35631 and the first AUG/CUG of ORF I is at about nt35532. Virtanen et al. (1984) J. Virol. 51: 822-831. Using any of theabove strategies for the other genes, a UP-TRE may be introducedupstream from the transcription start site. For the construction offull-length adenovirus with a hepatocellular carcinoma-specific TREinserted in the E4 region, the co-transfection and homologousrecombination are performed in W162 cells (Weinberg et al. (1983) Proc.Natl. Acad. Sci. 80:5383-5386) which provide E4 proteins in trans tocomplement defects in synthesis of these proteins.

Adenoviral constructs containing an E3 region can be generated whereinhomologous recombination between an E3-containing adenoviral plasmid,for example, BHGE3 (Microbix Biosystems Inc., Toronto) and anon-E3-containing adenoviral plasmid, is carried out.

Alternatively, an adenoviral vector comprising an E3 region can beintroduced into cells, for example 293 cells, along with an adenoviralconstruct or an adenoviral plasmid construct, where they can undergohomologous recombination to yield adenovirus containing an E3 region. Inthis case, the E3-containing adenoviral vector and the adenoviralconstruct or plasmid construct contain complementary regions ofadenovirus, for example, one contains the left-hand and the othercontains the right-hand region, with sufficient sequence overlap as toallow homologous recombination.

Alternatively, an E3-containing adenoviral vector of the invention canbe constructed using other conventional methods including standardrecombinant methods (e.g., using restriction nucleases and/or PCR),chemical synthesis, or a combination of any of these. Further, deletionsof portions of the E3 region can be created using standard techniques ofmolecular biology.

Insertion of an IRES into a vector is accomplished by methods andtechniques that are known in the art and described herein supra,including but not limited to, restriction enzyme digestion, ligation,and PCR. A DNA copy of an IRES can be obtained by chemical synthesis, orby making a cDNA copy of, for example, a picornavirus IRES. See, forexample, Duke et al. (1995) J. Virol. 66(3):1602-9) for a description ofthe EMCV IRES and Huez et al. (1998), Mol. Cell. Biol. 18(11):6178-90)for a description of the VEGF IRES. The internal translation initiationsequence is inserted into a vector genome at a site such that it liesupstream of a 5′ -distal coding region in a multicistronic mRNA. Forexample, in a preferred embodiment of an adenovirus vector in whichproduction of a bicistronic E1A-E1B mRNA is under the control of a livercancer-specific TRE, the E1B promoter is deleted or inactivated, and anIRES sequence is placed between E1A and E1B. In other embodiments, partor all of the 19-kDa region of E1B is deleted. IRES sequences ofcardioviruses and certain aphthoviruses contain an AUG codon at the 3′end of the IRES that serves as both a ribosome entry site and as atranslation initiation site. Accordingly, this type of IRES isintroduced into a vector so as to replace the translation initiationcodon of the protein whose translation it regulates. However, in an IRESof the entero/rhinovirus class, the AUG at the 3′ end of the IRES isused for ribosome entry only, and translation is initiated at the nextdownstream AUG codon. Accordingly, if an entero/rhinovirus IRES is usedin a vector for translational regulation of a downstream coding region,the AUG (or other translation initiation codon) of the downstream geneis retained in the vector construct.

Methods of packaging polynucleotides into adenovirus particles are knownin the art and are also described in co-owned PCT PCT/US98/04080.

Therapeutic Methods

An effective amount of the adenovirus vector is administered to apatient as a composition in a pharmaceutically acceptable excipient (andmay or may not be in the same compositions), including, but not limitedto, saline solutions, suitable buffers, preservatives, stabilizers, andmay be administered in conjunction with suitable agents such asantiemetics. An effective amount is an amount sufficient to effectbeneficial or desired results, including clinical results. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of an adenoviral vector is anamount that is sufficient to palliate, ameliorate, stabilize, reverse,slow or delay the progression of the disease state. Some individuals arerefractory to these treatments, and it is understood that the methodsencompass administration to these individuals. The amount to be givenwill be determined by the condition of the individual, the extent ofdisease, the route of administration, how many doses will beadministered, and the desired objective.

Delivery of adenoviral vectors is generally accomplished by eithersite-specific injection or intravenous injection. Site-specificinjections of vector may include, for example, injections into liverlesions, as well as intraperitoneal, intrapleural, intrathecal,intra-arterial, intra-tumor injections or topical application. Thesemethods are easily accommodated in treatments using the combination ofadenoviral vectors and chemotherapeutic agents.

The adenoviral vectors may be delivered to the target cell in a varietyof ways, including, but not limited to, liposomes, general transfectionmethods that are well known in the art (such as calcium phosphateprecipitation or electroporation), direct injection, and intravenousinfusion. The means of delivery will depend in large part on theparticular adenoviral vector (including its form) as well as the typeand location of the target cells (i.e., whether the cells are in vitroor in vivo).

If used as a packaged adenovirus, adenovirus vectors may be administeredin an appropriate physiologically acceptable carrier at a dose of about10⁴ to about 10¹⁴. The multiplicity of infection will generally be inthe range of about 0.001 to 100. If administered as a polynucleotideconstruct (i.e., not packaged as a virus) about 0.01 μg to about 1000 μgof an adenoviral vector can be administered. The adenoviral vector(s)may be administered one or more times, depending upon the intended useand the immune response potential of the host, and may also beadministered as multiple, simultaneous injections. If an immune responseis undesirable, the immune response may be diminished by employing avariety of immunosuppressants, or by employing a technique such as animmunoadsorption procedure (e.g., immunoapheresis) that removesadenovirus antibody from the blood, so as to permit repetitiveadministration, without a strong immune response. If packaged as anotherviral form, such as HSV, an amount to be administered is based onstandard knowledge about that particular virus (which is readilyobtainable from, for example, published literature) and can bedetermined empirically.

Embodiments of the present invention include methods for theadministration of combinations of a liver cancer-specific adenoviralvector and a second anti-neoplastic therapy, which may includeradiation, an anti-neoplastic agent, etc., to an individual withneoplasia, as detailed in co-owned U.S. application Ser. No. 09/814,357,expressly incorporated by reference herein. The chemotherapeutic agentand adenovirus may be administered simultaneously or sequentially, withvarious time intervals for sequential administration. In someembodiments, an effective amount of an adenoviral vector and aneffective amount of at least one antineoplastic agent are combined witha suitable excipient and/or buffer solutions and administeredsimultaneously from the same solution by any of the methods listedherein or those known in the art. This may be applicable when theantineoplastic agent does not compromise the viability and/or activityof the adenoviral vector itself.

Where more than one antineoplastic agent is administered, the agents maybe administered together in the same composition; sequentially in anyorder; or, alternatively, administered simultaneously in differentcompositions. If the agents are administered sequentially,administration may further comprise a time delay. Sequentialadministration may be in any order, and accordingly encompasses theadministration of an effective amount of an adenoviral vector first,followed by the administration of an effective amount of thechemotherapeutic agent. The interval between administration ofadenovirus and chemotherapeutic agent may be in terms of at least (or,alternatively, less than) minutes, hours, or days. Sequentialadministration also encompasses administration of a chosenantineoplastic agent followed by the administration of the adenoviralvector. The interval between administration may be in terms of at least(or, alternatively, less than) minutes, hours, or days.

Administration of the above-described methods may also include repeatdoses or courses of target-cell specific adenovirus and chemotherapeuticagent depending, inter alia, upon the individual's response and thecharacteristics of the individual's disease. Repeat doses may beundertaken immediately following the first course of treatment (i.e.,within one day), or after an interval of days, weeks or months toachieve and/or maintain suppression of tumor growth. A particular courseof treatment according to the above-described methods, for example,combined adenoviral and chemotherapy, may later be followed by a courseof combined radiation and adenoviral therapy.

Anti-neoplastic agents include those from each of the major classes ofchemotherapeutics, including but not limited to: alkylating agents,alkaloids, antimetabolites, anti-tumor antibiotics, nitrosoureas,hormonal agonists/antagonists and analogs, immunomodulators,photosensitizers, enzymes and others. In some embodiments, theantineoplastic is an alkaloid, an antimetabolite, an antibiotic or analkylating agent. In certain embodiments the antineoplastic agentsinclude, for example, thiotepa, interferon alpha-2a, and the M-VACcombination (methotrexate-vinblastine, doxorubicin, cyclophosphamide).Preferred antineoplastic agents include, for example, 5-fluorouracil,cisplatin, 5-azacytidine, and gemcitabine. Particularly preferredembodiments include, but are not limited to, 5-fluorouracil gemcitabine,doxorubicin, miroxantrone, mitomycin, dacarbazine, carmustine,vinblastine, lomustine, tamoxifen, docetaxel, paclitaxel or cisplatin.The specific choice of both the chemotherapeutic agent(s) is dependentupon, inter alia, the characteristics of the disease to be treated.These characteristics include, but are not limited to, location of thetumor, stage of the disease and the individual's response to previoustreatments, if any.

In addition to the use of single antineoplastic agents in combinationwith a particular adenoviral vector, the invention also includes the useof more than one agent in conjunction with an adenoviral vector. Thesecombinations of antineoplastics when used to treat neoplasia are oftenreferred to as combination chemotherapy and are often part of a combinedmodality treatment which may also include surgery and/or radiation,depending on the characteristics of an individual's cancer. It iscontemplated that the combined adenoviral/chemotherapy of the presentinvention can also be used as part of a combined modality treatmentprogram.

There are a variety of delivery methods for the administration ofantineoplastic agents, which are well known in the art, including oraland parenteral methods. There are a number of drawbacks to oraladministration for a large number of antineoplastic agents, includinglow bioavailability, irritation of the digestive tract and the necessityof remembering to administer complicated combinations of drugs. Themajority of parenteral administration of antineoplastic agents isintravenously, as intramuscular and subcutaneous injection often leadsto irritation or damage to the tissue. Regional variations of parenteralinjections include intra-arterial, intravesical, intra-tumor,intrathecal, intrapieural, intraperitoneal and intracavity injections.

Delivery methods for chemotherapeutic agents include intravenous,intraparenteral and introperitoneal methods as well as oraladministration. Intravenous methods also include delivery through a veinof the extremities as well as including more site specific delivery,such as an intravenous drip into the portal vein of the liver. Otherintraparenteral methods of delivery include direct injections of anantineoplastic solution, for example, subcutaneously, intracavity orintra-tumor.

Assessment of the efficacy of a particular treatment regimen may bedetermined by any of the techniques known in the art, includingdiagnostic methods such as imaging techniques, analysis of serum tumormarkers, biopsy, the presence, absence or amelioration of tumorassociated symptoms. It will be understood that a given treatment regimemay be modified, as appropriate, to maximize efficacy.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

Using a hepatocellular carcinoma (HCC) mouse model, it has been shownthat the CRG-L2 gene is specifically expressed in the HCC tissue, butnot normal liver tissue. In this model, in situ hybridization showedthat CRG-L2 mRNA levels increased during early development of HCC (seeGraveel et al. (2003) Oncogene 22:1730-1736). In humans, CRG-L2 mRNA isupregulated in HCC tissues but displays restricted expression in normaltissues, except for the placenta. Based on this discovery, CGI initiateda research collaboration to clone the CRG-L2 TRE.

In order to characterize the CRG-L2 transcriptional regulatory elements,a series of cloned DNA fragments upstream of the CRG-L2 gene wereanalyzed. A 0.7 kb DNA fragment from the upstream region of the CRG-L2gene was shown to have promoter activity in HCC cell lines (FIG. 1). Theconstruct CP1505 (CRG-L2 0.7 kb), pGL2 (promoter-less luciferaseconstruct), and AFP luciferase construct were transfected into Hep3B andHuH7 cells. After 72 hours, cells were harvested and assayed forluciferase activity. Data presented in FIG. 3 showed that both Hep3B andHuH7 cells transfected with CP1505 produced a significant luciferaseactivity. In contrast, the luciferase activity detected in HBL-100 cells(FIG. 4) is eight to ten fold less than the activity seen in Hep3B andHuH7 cells, demonstrating cell specificity.

It is evident from the above results that adenoviruses can be developedwith specificity for particular host cells, where the viruses arereplication-competent. The viruses may be vehicles for the introductionof a wide variety of genes into particular target cells.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1. A replication-competent adenovirus vector comprising an adenovirusgene essential for replication under transcriptional control of ahepatocellular carcinoma specific TRE, wherein said TRE consistsessentially of a CRG-L2 TRE selected from the group consisting of SEQ IDNO: 1 and nucleotides 119-803 of SEQ ID NO: 1, and wherein the TREprovides for expression of the adenovirus gene essential for replicationin hepatocellular carcinoma cells expressing CRG-L2.
 2. The adenovirusvector according to claim 1,wherein said CRG-L2 TRE comprises a humanpromoter or enhancer.
 3. The adenovirus vector according to claim 1further comprising a second human transcriptional regulatory element. 4.The adenovirus vector according to claim 1,wherein said hepatocellularcarcinoma cell specific TRE comprises two or more enhancers.
 5. Theadenovirus vector according to claim 1,wherein the adenoviral vectorfurther comprises a second adenovirus gene co-transcribed undertranscriptional control of the TRE.
 6. The adenovirus vector accordingto claim 5, wherein the second adenovirus gene is under translationalcontrol of an IRES.
 7. The adenovirus vector of claim 1,wherein saidadenoviral gene essential for replication is E1A or E1B.
 8. Theadenovirus vector of claim 7, wherein E1A or E1B has a mutation in ordeletion of its endogenous promoter.
 9. The adenovirus vector of claim7, wherein the E 1B gene's 19-kDa region is deleted.
 10. A compositioncomprising: the replication-competent adenovirus vector according toclaim 1 and a pharmaceutically acceptable excipient.
 11. A host cellline comprising the adenovirus vector of claim 1.